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became broadly available, isolators were made from
stainless steel and were large, heavy and difficult to
handle. The first stainless steel isolators were developed
by Reyniers and Trexler and were designed as autoclave
chambers. Rigid steel isolators with steam-sterilized
locks were also developed by Gustafsson and were
used for decades. In the late 1950s, Trexler designed
the first transparent and flexible-film plastic polyvinyl
chloride (PVC) isolator.20 These types of isolator as
well as semi-rigid isolators are commonly used today.
Isolators are designed as closed, impermeable barriers with a sterile inner environment. They can be
rigid- or flexible-wall isolators, and the material most
commonly used today is clear PVC. Additionally, in
gnotobiotic husbandry air pressure within an isolator
is kept positive. This can prevent the entry of microorganisms through microscopic punctures on the isolator, but it also keeps a flexible-film isolator inflated
without the need for a frame to support it. The main
components of isolator construction have not changed
much in the last 50 years, and include an isolation
chamber, a port system, an air inlet and outlet filter,
a blower that maintains positive pressure by pushing air
into the isolator chamber and arm-length gloves.
Complete gnotobiotic husbandry of gnotobiotic animals is performed without direct contact between the
outside environment and the animals. The critical parts
of the isolator are the gloves and the transfer port. All
procedures in the isolator are performed through armlength gloves. The gloves can be made from different
materials such as nitrile rubber, latex or polyurethane.11 Furthermore, it is very important to inspect the
gloves for any damage before each use to prevent possible contamination. The second critical point is the
transfer port, which connects the sterile isolator chamber to the outside environment. The port is usually
designed as a plastic or stainless-steel cylinder bordered
by double doors.11 Isolators fitted with a single-door
port are also available; however, these isolators must
be connected to a transfer isolator or specific cylinder
for transferring materials from and into these isolators.
All materials needed for animal maintenance as well as
for the experimental setup are introduced into the isolator through the transfer port. Supplies are usually
introduced by using a supply cylinder made from stainless steel and wrapped in a filter medium that allows
steam penetration during the autoclaving cycle.11 The
cylinders are loaded with autoclave-resistant supplies
such as bedding, food or nesting material, closed with
biaxially-oriented polyethylene terephthalate film (such
as MylarÕ or HostaphanÕ ) and autoclaved.11
Autoclaving protocols need to be optimized in each
facility and adapted to particular needs and material
properties.21 Furthermore, all sterilization processes,
including the autoclave cycles, need to be validated.

Laboratory Animals 53(3)
After sterilization is confirmed, the cylinder can be
docked onto the isolator port by using a PVC transfer
sleeve that was previously sterilized by a highly effective
chemical sterilant (such as peracetic acid, chlorine dioxide or 10% bleach)22 to create a sterile tunnel between
the cylinder and the isolation chamber. In our facility,
2% buffered peracetic acid is used as the main sterilant.
Isolator housing is optimal for long-term maintenance and breeding of gnotobiotic rodent colonies.
Furthermore, it is also advisable to perform long-term
gnotobiotic experiments in the isolators. However,
because a single isolator represents one hygienic unit,
it is only possible to keep experimental groups identical
in their microbiological status within one isolator.
Furthermore, because isolators offer open-cage housing, an introduced contaminant will rapidly spread to
all cages. Moreover, at the end of the study or upon
contamination the entire isolator needs to be disassembled, sterilized and made germ-free again. This procedure is time consuming and expensive. Therefore,
several systems for short-term gnotobiotic rodent housing have been developed. These systems include static
gnotocages such as Hannover gnotocages (Han-gnotocages), or airtight sealed individually-ventilated cage
(IVC) systems such as the Tecniplast IsoCage P
system. This cage technology represents a mini isolator
at a cage level and allows animal handling in a class II
biosafety cabinet.21,23 Nevertheless, the sterile handling
procedure requires well-trained staff and strict adherence to the operating procedures to maintain sterility or
unchanged microbiological status of the gnotobiotic
animals. In our laboratory, the operating procedure
involves cold sterilization of the biosafety cabinet and
cage surfaces with an effective sterilant. The work is
performed by two persons: a 'sterile' partner and a
'semi-sterile' partner. Both persons wear personal protective equipment including a sterile coat, sterile surgical gloves, a face mask and a hair bonnet. The semisterile partner is responsible for opening the cage and
assists the sterile partner by providing sterile materials.
The sterile partner is only allowed to touch items within
the cage and sterile materials provided by the semi-sterile partner. However, due to the handling of materials
and animals within the biosafety cabinet, the risk of
contamination is higher than in the isolator.
Moreover, each cage represents a separate hygienic
unit. Therefore, these techniques enable work with multiple different gnotobiotic groups simultaneously with
separation at the cage level. The Han-gnotocage is built
from autoclavable rigid plastic sealed with a metal lid
filled with a filter medium (Figure 1(b)). Air flow
through the filter medium occurs naturally and is not
driven by a ventilation system. In the isocage-system,
the air flow is driven mechanically and enters the cage
by passing through a high-efficiency particulate air



Laboratory Animals - June Issue

Table of Contents for the Digital Edition of Laboratory Animals - June Issue

Contents
Laboratory Animals - June Issue - Cover1
Laboratory Animals - June Issue - Cover2
Laboratory Animals - June Issue - Contents
Laboratory Animals - June Issue - 214
Laboratory Animals - June Issue - 215
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Laboratory Animals - June Issue - Cover3
Laboratory Animals - June Issue - Cover4
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